A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

Summary

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Abstract

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the “gold standard” enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

Introduction

When analyzing complex clinical or environmental samples (e.g., feces, soils), there are two main methodologies used for the extraction of DNA. The first is a mechanical disruption of the matrix using an intense bead-beating step, while the second is an enzymatic disruption of the matrix to chemically release bacterial cells and inhibit PCR inhibitors from the matrix simultaneously. Given the different means by which these two types of extraction methods work, it is not surprising that previous studies demonstrated that the appropriate DNA extraction method is both highly sample and analysis dependent. Comparative DNA extraction studies previously showed that some methods are more appropriate for improved DNA quality and quantity from environmental samples^1-3, while others demonstrated improvements for community-level analyses such as denaturing gradient gel electrophoresis (DGGE)^4-6, terminal restriction fragment length polymorphism (T-RFLP)⁷, automated ribosomal intergenic spacer analysis (ARISA)⁸, and phylogenetic microarrays⁹. Therefore, appropriate DNA extraction methods need to be used, or developed, according to the types of environmental samples and the types of analyses being performed on those samples, especially given the recent advancements in bacterial community analyses.

Next generation sequencing, in conjunction with more quantitative community assessments (e.g., quantitative PCR (qPCR)), is becoming more prevalent in the environmental and clinical sciences, however, very little research has been performed to determine the effect of DNA extraction methods on these data sets. Most DNA extraction comparison studies dealt with microbiomic community estimates from human or human model samples^10,11, not agricultural animal samples. The few poultry-focused next generation sequencing studies dealt with specific metagenomic^12,13 or microbiomic¹⁴ questions; they did not discuss the effect of DNA extraction method on the resulting microbiomic analyses. Considering the complex nature of environmental samples related to poultry production (e.g., feces, litter/bedding, pasture soil), DNA extraction methods need to be carefully selected. Poultry-related environmental samples are known to contain large numbers of PCR inhibitors and up to 500-fold DNA extract dilutions have been required for PCR and subsequent downstream analysis^15-17. Therefore it is essential that DNA extraction methods be optimized for these types of samples in order to not only physically disrupt the matrix, but also to be able to reduce/eliminate the large number of inhibitors that are present.

The QIAamp DNA Stool Mini Kit, an enzymatic extraction method, has been considered the “gold standard” when extracting DNA from difficult gut/fecal samples^1,18,19 and has been applied successfully to poultry environmental samples^8,14. The enzymatic removal of PCR inhibitors through the use of a proprietary matrix is one of the greatest advantages of using this method for these types of environmental samples, as is the ability to significantly improve throughput (and reduce sample processing error) using automated workstations. One major disadvantage is the lack of a mechanical homogenization step to physically disassociate bacterial cells from the environmental matrix. When testing gut and fecal samples of non-poultry origin, the addition of a bead-beating or mechanical disruption step within a DNA extraction protocol significantly increased extraction efficiency⁹, DNA yield/quality^1,4,5 and significantly improved downstream community analyses in terms of richness, diversity, and coverage^5,6,11. These studies compared not only mechanical bead-beating methods to the “gold standard” enzymatic method, but some also added the mechanical bead-beating step to the enzymatic protocol to improve results^6,9,11.

According to the results from the above studies, bacterial community analyses (both qualitative and quantitative) could be improved from poultry-related environmental samples through the addition of a mechanical homogenization step to the enzymatic method. Therefore, the goal of this study was twofold: (1) to develop a novel DNA extraction technique that utilizes the most desirable aspects of both the mechanical (powerful homogenization step) and enzymatic (PCR inhibitor removal and automation) extraction methods and (2) compare the quantitative (via qPCR) and qualitative (via microbiomics) bacterial community assessments of this novel method to representative mechanical and enzymatic methods.

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Protocol

1. Mechanical Homogenization of Environmental Poultry Production Samples

1. Prior to extraction, set a water bath to 95 °C and allow the water bath time to reach that temperature.
2. Weigh out 0.33 g of soil or fecal material into a 2 ml Lysing Matrix E tube.
   1. Do not exceed 0.33 g of sample in the tube, since this will cause the following solutions to exceed the capacity of the tube.
   2. Thaw frozen samples to RT prior to weighing.
   3. In order to analyze a total of 1 g of soil/feces, weigh out 3 replicate 0.33 g samples for each individual environmental sample.
   4. Store samples at -20 °C within the conical matrix tubes prior to extraction, if needed.
3. Add 825 µl Sodium Phosphate Buffer and 275 µl of PLS solution to a sample tube. Mix using a vortex for ~15 sec and then centrifuge the samples at 14,000 x g for 5 min.
4. Decant the supernatant and add 700 µl of Buffer ASL. Mix using a vortex for 5 sec.
   1. Ensure that there is headspace (~10% total volume) available in the conical tube at this point. If there is no headspace, the tubes will have a tendency to leak during the next homogenization step which could lead to cross-contamination and/or sample loss.
5. Place the samples into a FastPrep 24 Instrument, and homogenize the samples at a speed of 6.0 m/sec for 40 sec.
6. Centrifuge the homogenized sample at 14,000 x g for 5 min. Transfer the supernatant to a sterile 2 ml microcentrifuge tube.
7. To maximize the DNA recovery from the sample, repeat steps 1.4 to 1.6, combining the supernatants into the same sterile, 2 ml microcentrifuge tube.

2. Enzymatic Inhibition of Inhibitors from Sample Homogenates

NOTE: This protocol uses the QIAamp DNA Stool Mini Kit.

1. Incubate the supernatant in a 95 °C water bath for 5 min to maximize DNA recovery from any remaining cells within the supernatant.
   1. Incubate at 70 °C for samples containing mostly Gram-negative organisms. However if Gram-positive organisms are present (which is the case with poultry fecal samples), incubate at 95 °C.
   2. Use plastic locking clips on the microcentrifuge tubes to ensure that the tubes will not “pop” open and potentially lose sample volume as pressure can build up in these sealed microcentrifuge tubes.
2. Open each microcentrifuge tube to release the pressure, re-cap the microcentrifuge tubes and mix using a vortex for 15 sec.
3. Centrifuge the sample at 14,000 x g for 1 min, remove 1.2 ml of the supernatant and place it into a new sterile 2 ml microcentrifuge tube.
4. Add 1 InhibitEx tab to each sample, and mix using a vortex until the sample becomes a uniformly white/off-white liquid.
   1. Avoid touching the InhibitEx tab while placing it into the microcentrifuge tube containing the sample. To accomplish this, place the blister pack containing the tab directly over the open microcentrifuge tube and gently push the tab out of the blister pack and into the microcentrifuge tube.
5. Incubate the sample for 1 min at RT (~ 25 °C) and centrifuge at 14,000 x g for 5 min.
6. Transfer all liquid to a sterile 1.5 ml microcentrifuge tube and centrifuge at 14,000 x g for 5 min.
   1. Avoid any remaining particulates that may have pelleted at the bottom of the microcentrifuge tube at the end of step 2.5 when transferring the liquid.

3. Automated DNA Purification Using the QIAcube Robotic Workstation

NOTE: The number of plastic consumables, the arrangement of the sample rotor adapters within the centrifuge, and the required volumes of the buffers/solutions are dependent on the number of samples that are being run.

1. Add elution tubes and filter tubes to the appropriate slots within the rotor adapters. For each sample, add 400 µl to the middle slot of the rotor adapter. Place the rotor adapters in the workstation centrifuge in the correct arrangement according to the number of samples being purified.
   1. Make sure that all of the microcentrifuge tube lids are properly secured within the rotor adapter since a failure to do so could result in shearing during one of the centrifugation steps of the purification protocol.
2. Add the required number of 1,000 µl and 200 µl filter-tips to the workstation, and fill the supplied buffer bottles with the required volume of buffers.
   NOTE: The buffers required for this purification protocol (AL, AW1, AW2, and AE) are all contained within the QIAamp DNA Stool Mini Kit. The user needs to supply the 100% ethanol that is needed for the AW buffers and as a solution used in the purification process.
3. Add the required volume of the supplied proteinase K solution into a sterile 1.5 ml microcentrifuge tube and place it into slot A on the workstation. Also, add the required number (equal to the number of samples being purified) of 2 ml safe-lock microcentrifuge sample tubes RB to the shaker section of the workstation.
   1. Ensure that the lids of the sample tubes are securely placed into the appropriate slots on the workstation, since a failure to do so will result in an error when the machine initially scans the workstation to make sure all needed plastics and liquids are available for the requested run.
4. Using the touchscreen on the workstation, select the DNA Stool – Human Stool – Pathogen Detection Protocol, and read through the subsequent screens to ensure that the workstation was loaded correctly. Once all check screens are passed, select Start to run this protocol.
   1. If extracting DNA from more than 12 samples, begin the homogenization process (Step 1) for the next set of samples, since a run of 12 samples takes ~72 min to complete on the workstation.
5. Remove the samples from the rotor adapters, cap them, and place at -20 °C until needed for subsequent downstream analyses.
   1. At this point, combine the 3 replicate purifications for an individual sample (total analyzed amount = 1 g) using a centrifugation/evaporation-based system. Combine the replicates and re-elute to a final volume of 100 µl of Tris-ETDA buffer.

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Results

For this study, fresh fecal droppings and litter samples were recovered from a commercial broiler house (~25,000 birds) in the southeastern US. The broilers (Gallus gallus) were Cobb-500 crosses, and they were 59 days old at the time of sampling. Fresh fecal and litter samples were recovered from four distinct areas within the house (near cooling pad, near the waterer/feeder lines, in between the waterer/feeder lines, and near exhaust fans), and samples from each of these areas contained five pooled samples from...

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Discussion

The DNA extraction method used effected the quantitative and qualitative total bacterial community estimates for both the fecal and litter samples, supporting the sample analyses dependent nature of DNA extraction methods seen previously^1,3,6. For both the fecal and litter samples, the order of performance of the DNA extraction methods was different for the quantitative (Mechanical > Hybrid > Enzymatic) and the qualitative (Enzymatic > Hybrid > Mechanical) total bacterial community estimates. W...

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Materials

Name	Company	Catalog Number	Comments
Lysing Matrix E tube	MPBio	6914-050	Different sizes available and the last 3 numbers of the cat. No. indicate size (-050 = 50 tubes, -200 = 200 tubes, -1000 = 1,000 tubes)
Sodium Phosphate Solution	MPBio	6570-205	Can be purchased individually, or also contained within the FastDNA Spin Kit for feces (Cat. No. 116570200)
PLS Buffer	MPBio	6570-201
Buffer ASL (560 ml)	Qiagen	19082
FastPrep 24 homogenizer	MPBio	116004500	48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step
QIAamp DNA Stool Mini Kit	Qiagen	51504
QIAcube24 (110V)	Qiagen	9001292	Preliminary results show that QIAcube HT (Cat. No. 9001793) can be used to improve throughput, but different consumables are required of this machine and more comparative work needs to be done.
Filter-Tips, 1,000 ml (1024)	Qiagen	990352
Filter-Tips, 200 ml (1024)	Qiagen	990332
QIAcube Rotor Adapters (10 x 24)	Qiagen	990394	For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative.  It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690
Sample Tubes RB (2 ml)	Qiagen	990381	Alternative: Eppendorf Safe-Lok micro test tube, Cat. No. 022363352